iTRAQ-Based Shotgun Proteomics Approach for Relative Protein Quantification
Shotgun proteomics has a key role in quantitative estimation of proteins from biological systems under different conditions, which is crucial in the understanding of their functional roles. Isobaric tagging for relative and absolute quantitation (iTRAQ) m
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Introduction Shotgun proteomics is no longer merely descriptive, but has become quantitative [1]. The estimation of protein abundances in different biological conditions provides more information than in qualitative studies, in which only the presence or posttranslational modifications of proteins are determined. One of the turning points in quantitative proteomics was the implementation of isobaric methods for relative protein quantification. Different from other stable isotope labeling methods in which identical peptides with a mass variance from two or more samples can be distinguished in the mass spectrometry (MS) 1 spectrum, isobaric methods add isotope tags of the same mass to each peptide. In MS1 mode, each peptide labeled with different isobaric tags is undistinguishable but in MS2 mode reporter ions of distinct masses are released such that peptides from different samples can be differentiated and quantified, based on the intensity of these reporter ions [2]. Two of the most used isobaric labeling methods are isobaric
Paul C. Guest (ed.), Multiplex Biomarker Techniques: Methods and Applications, Methods in Molecular Biology, vol. 1546, DOI 10.1007/978-1-4939-6730-8_23, © Springer Science+Business Media LLC 2017
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tags for relative and absolute quantitation (iTRAQ) [3] and tandem mass tags (TMT) [4, 5]. The widely employed iTRAQ approach is a reliable labeling technique employed in the analysis of up to four (iTRAQ 4-plex) [3] or eight samples (iTRAQ 8-plex) [6]. Compared to label-free mass spectrometry techniques, the multiplexing potential of isotopic labeling increases the statistical relevance and accuracy of results through the simultaneous analysis of different biological samples and through normalization to an internal standard. In the iTRAQ 4-plex approach, the reagent contains reporter (N-methylpiperazine), balance (carbonyl) and reactive groups (NHS ester). Each reagent has the same mass achieved by a combination of 13C, 15 N, and 18O in the reporter (m/z 114-117) and balance groups (28– 31 Da). The labeled peptides have identical retention times in liquid chromatography and, since the tags are isobaric, the peptides appear as single peak with the same m/z in a MS1 spectrum. However, selection of the precursor ion for fragmentation produces a MS2 spectrum with reporter ion peaks at low mass region (114, 115, 116, and 117) and peptide backbone fragmentation peaks. Intensity of the reporter ion peaks directly reflects the abundance of the peptide in each sample (Fig. 1). Here, we describe a detailed sample preparation and iTRAQ 4-plex labeling method for relative quantification of multiple samples from human and plant tissues. Additionally, we detail two strategies for peptide fractionation after iTRAQ labeling.
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Materials (See Note 1)
2.1 Protein Extraction of Human Tissues 2.2 Protein Extraction of Plant Tissues
1. Human tissues: RapiGest® (Waters Corporation). 2. 1 M triethylammonium bicarbonate (TEAB; Sigma-Aldrich) (see Note 2). 1. 50 mM pyridine (pH
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